The present invention relates to the identification of HD-2, a natural chemical substance that was separated and purified from a natural product, Sinsuk, as arsenic hexoxide (As4O6) and about its therapeutic efficacy as an anti-cancer drug and pharmaceutical composition, and more particularly, to the purification processes of a natural chemical substance (arsenic hexoxide, As4O6) from Sinsuk while eliminating the toxicity and the novel anti-cancer effect of As4O6 and its pharmaceutical composition by their direct cytotoxicity and suppression of new angiogenesis at and around tumor sites.
In general, various drugs are presently available for anti-cancer chemotherapy. Alkylating agents, such as cisplatin and cyclophosphamide, manifest their anti-cancer effect by forming covalent bonds with nitrogen atoms of DNA nucleotides because of its highly electrophilic property of the active site. Antimetabolites, such as 5-fluorouracil, act by inhibiting enzymes involved in biosynthesis of nucleic acids or by being inserted into DNA or RNA structures by itself. Some antibiotics, such as adriamycin, act potently on DNA to inhibit the normal function, which results in suppression of tumor growth. But all of these anti-cancer agents affect not only pathological tumor cells, but also normal healthy cells, especially bone marrow cells or intestinal epithelia with high turnover rate, which cause serious complications and toxicity, such as myelosuppression, alopecia, renal failure, nausea and vomiting, neurotoxicity, etc.
On the other hand, arsenic has been known as a potent, environmental carcinogen, affecting skin and lung often. Arsenic is reported to bind to sulfohydryl structure of enzymes to inactivate target enzymes, to inhibit phosphorylation and dephosphorylation reactions, which are vital for regulation of enzyme activities, and to cause abnormalities in chromosomes. Therefore arsenic has been studied mostly from toxicological aspect, related to these reports up to now.
But in the past, arsenic had been used as a therapeutic agent in both Oriental and Occidental medicines. Especially, in traditional Chinese medicine including Korean medicine, arsenic compound had been prescribed for a long time to treat some fatal diseases, e.g. to eradicate evil energy. In old medical literatures of Korea and China, it is described that arsenic was prescribed as a medicine by the name of Eungwhang in page 1234 or by the name of Bisang in page 1237 of TonEuiBoGam (NamSaDang), or Encyclopedia of Oriental Medicine, where it is described that arsenic was prescribed only after reducing its toxicity, because of its extreme toxicity. Also arsenic was known to have detoxifying activity against several toxic substances. For example, arsenic was used in managing choongak, or vomiting and in eradicating spirits and evil energy. In an old literature of Chinese medicine (BonChoKangMok (Encyclopedia of Herbs of Chinese Medicine), pages 12-16 of vol. 9), indications and pharmacological actions of arsenic (by the name of whangwoong) are described, where arsenic is reported to have the action of purifying the blood. Thus arsenic had been recognized as an active medicine and used for a long time, but in Korea, arsenic is recognized as a possibly harmful chemical with characteristics of heavy metals and accordingly its use is quite limited. Arsenic possesses some characteristics of heavy metals although it does not belong to heavy metal group and therefore, has been avoided in the production of medicine. Exposure to arsenic leads to anemia, leukopenia, and dysfunction of kidney and liver and chronic exposure may have carcinogenic effect.
In Western medicine, arsenic compound was prescribed for treating several diseases, including rheumatism, syphilis, psoriasis, etc. and low dose of arsenic compound had been known to have beneficial effect on physiological functions of human body, including stimulation of hematopoiesis, which coincides with descriptions in old literatures of Oriental medicine. But in modern medicine, indications for arsenic compound became very limited. From the end of 19th century to the beginning of 20th century, arsenic compound was tried to treat chronic leukemia and after 1950s, melarsoprol, an organic compound of arsenic which was prescribed for African trypanosomiasis, is the only arsenic compound in use at present time.
Based on these pharmacological properties of arsenic, attempts have been made recently to develop a novel anticancer drug and presently some studies are making rapid progress in this field. After the Cultural Revolution, China has been putting considerable efforts to study traditional medicine using the scientific tools of Western medicine. They published a report in 1996, in collaboration with a French research team, that arsenic trioxide (As2O3) had an excellent effect in treating acute promyelocytic leukemia. Researchers of Western medicine were marveled at this result, because arsenic trioxide was especially effective in treating leukemia patients who had been resistant to conventional chemotherapy since this paper was published, more medical scientists of Western Hemisphere became interested in the possible anticancer effect of arsenic compounds. Stimulated by these results, considerable efforts have been made to integrate traditional Oriental Medicine and modern molecular medicine by interpreting the results of Oriental Medicine in terms of mainstream modern anticancer chemotherapy. It is extremely important to develop novel chemicals to have effective anticancer efficacy without any serious side effects. The invention described here succeeded in the separation and purification of the active ingredient by treating a natural, raw material of arsenic, which had been used in Oriental medicine, through multiple processes. Additionally clinical study indicated that pharmaceutical composition of arsenic hexoxide shows potent anticancer efficacy without any obvious side effects.
Accordingly, the present invention is to provide a novel, natural chemical substance, arsenic hexoxide (As4O6) obtained from Sinsuk, while eliminating toxicity.
The other object of this invention to elucidate the action mechanism of anticancer efficacy of the novel, natural chemical substance obtained from Sinsuk.
Another object of the invention is to describe the usage of the novel, natural chemical substance for anticancer therapy and its pharmacological composition.
To achieve these advantages in accordance with the purpose of the present invention, as embodied and broadly described, first we separated and purified a natural chemical substance, HD-2, by repeated heating of Sinsuk containing arsenic to eliminate the toxicity, which was followed by structure analysis. The white substance obtained by this procedure was tested on cloned tumor cells of mice and human beings, to evaluate the anticancer efficacy of the substance and to see whether the anticancer effects are caused by tumor cell death by apoptosis mechanism. Toxicity of HD-2 following acute administration was evaluated by observing clinical symptoms of rats after a single, oral administration of a large dose and The toxicity of HD-2 following subacute administration was evaluated by observing clinical symptoms of rats after a slow oral administration. Clonal tumor cells, targeted to lungs, were injected intravenously into mice and HD-2 was administered orally or through intravenous route. Afterward the number of metastatic tumor masses in Lungs was counted to evaluate the inhibitory effect of the substance on cancer metastasis. Similarly, melanoma cells were inoculated intradermally into mice, followed by oral administration of HD-2, after which anticancer mechanism of HD-2 was investigated by counting the number of new blood vessels formed at or around tumor masses. Cancer was induced by injection of carcinogen into mice and tumor-suppressing efficacy was measured in these mice after oral administration of HD-2. We also tested a pharmacological composition prepared by mixing various herbs of Chinese medicine with arsenic hexoxide, which was administered orally to cancer patients at terminal stage to evaluate the anticancer efficacy.
In accordnace with one aspect, the present invention provides an anti-cancer agent of arsenic hexoxide (As4O6) of a natural chemical substance and its pharmaceutical composition comprising:
1) We separated and purified a natural chemical substance of white color, HD-2, by repeated heating of Sinsuk containing arsenic and arsenic of reagent grade, which were followed by structure analysis to show that it corresponds to arsenic hexoxide, As4O6.
2) A natural chemical substance, As4O6, obtained by this procedure was added to culture media to grow cloned tumor cells of mice and human beings, to evaluate the anticancer efficacy of the substance.
3) The anticancer mechanism of As4O6 was studied to examine whether the anticancer efficacy was due to tumor cell death by apoptosis mechanism.
4) Different amounts of a natural chemical substance, As4O6, was acutely administered orally to male and female rats, to examine the acute toxicity of arsenic hexoxide by observing manifested complications.
5) Same amount of a natural chemical substance, As4O6, was slowly administered orally to male and female rats, to examine the subacute toxicity of the invention by observing manifested complications.
6) Clonal tumor cells, targeted to lungs, were injected intravenously into mice and a natural chemical substance, As4O6, was administered orally or through intravenous route. Afterwards the number of metastatic tumor masses appearing in lungs was counted to evaluate the inhibitory effect of the substance on cancer metastasis.
7) Similarly, malignant melanoma cells were inoculated intradermally into mice followed by oral administration of a natural anticancer agent, As4O6. Afterwards anticancer mechanism was studied by measuring the size of tumor masses and by counting the number of newly formed blood vessels at and around tumor masses.
8) Carcinogen was injected into mice to induce malignant tumors and anticancer effects of a natural anticancer agent, As4O6, were studied by measuring the incidence and size of tumors in liver and lung.
9) We also prepared pharmaceutical composition by adding various herbs of Oriental medicine to a natural anticancer agent, As4O6, in several forms (tablet, capsule, and solution).
10) Tablets prepared as described above were administered orally to cancer patients at terminal stage, carrying a malignant cancer of uterus, lung, maxillary sinus, kidney, or urinary bladder, to evaluate the therapeutic efficacy of As4O6. The size of the tumors and clinical courses were monitored using computed tomography (CT) and magnetic resonance imaging (MRI).
It is to be understand that both the forgoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.